Osteoporosis is a leading cause of disability in the elderly, particularly elderly women. It has recently been realized that human parathyroid hormone (hPTH) and certain analogues are stimulators of bone growth that are useful in the treatment of osteoporosis. Osteoporosis is a progressive disease which results in the reduction of total bone mass and increased bone fragility. This often results in spontaneous fractures of load-bearing bones and the physical and mental deterioration characteristic of immobilizing injuries. Postmenopausal osteoporosis is caused by the disappearance of estrogens which trigger a decade-long acceleration of bone turnover with an increased imbalance between resorption of old bone and formation of new bone. This results in thinning, increased porosity, and trabecular depletion of load-bearing bones. Osteoporosis is also associated with hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and the use of certain steroidal drugs. Remedies historically have involved increase in dietary calcium, estrogen therapy, and increased doses of vitamin D, but mainly with agents such as antiresorptives that inhibit bone resorption by osteoclasts.
Parathyroid hormone (PTH) is produced by the parathyroid gland and is a major regulator of blood calcium levels. PTH is a polypeptide and synthetic polypeptides may be prepared by the method disclosed by Erickson and Merrifield , The Proteins, Neurath et al., Eds., Academic Press, N.Y., 1976, page 257, and as modified by the method of Hodges et al (1988) Peptide Research 1, 19 or by Atherton, E. And Sheppard, R. C. Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989.
When serum calcium is reduced to below a normal level, the parathyroid gland releases PTH and the calcium level is increased by resorption of bone calcium, by increased absorption of calcium in from the intestine, and by increased renal reabsorption of calcium front nascent urine in the kidney tubules. Although continuously infused low levels of PTH can remove calcium from the bone, the same low doses, when intermittently injected can actually promote bone growth.
Tregear, U.S. Pat. No. 4,086,196, described human PTH analogues and claimed that the first 27 to 34 amino acids are the most effective in terms of the stimulation of adenylyl cyclase in an in vitro cell assay. Rosenblatt, U.S. Pat. No. 4,771,124, disclosed the property of hPTH analogues wherein Trp.sup.23 is substituted by amino acids phenylalanine, leucine, norleucine, valine, tyrosine, .beta.-napthylalanine, or .alpha.-napthylalanine as a PTH antagonist. These modified hPTH analogues also have the 2 and 6 amino terminal acids removed, resulting in loss of most agonist activities when used to treat osteoporosis. These analogues were designed as inhibitors of PTH and PTH-related peptide. The analogues were claimed as possibly useful in the treatment of hypercalcemia associated with some tumors.
Pang et al, WO93/06845, published Apr. 15, 1993, described analogues of hPTH which involve substitutions of Arg.sup.25, Lys.sup.26, Lys.sup.27 with numerous amino acids, including alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. These are claimed, with no supporting data from animal or human trials, to be effective in the treatment of osteoporosis with minimal effects on blood pressure and smooth muscle.
Other references of interest which disclose hPTH analogues are PCT published application no. WO9851324, of Condon and Morze, U.S. Pat. No. 5,747,456 of Chorev et al., and U.S. Pat. No. 5,717,062 of Rosenblatt et al., which disclose some single and double cyclics involving 13-17 and 26-30 cyclizations.
PTH operates through activation of two second messenger systems, G.sub.s -protein activated adenylyl cyclase (AC) and G.sub.q -protein activated phospholipase C.sub..beta.. The latter results in a stimulation of membrane-bound protein kinase Cs (PKC) activity. In a rat osteosarcoma(RCO) cell line the PKC activity has been shown to require PTH residues 29 to 32 (Jouishomme et al (1994) J. Bone Mineral Res. 9, (1179-1189). It has been established that the increase in bone growth, i.e. that effect which is useful in the treatment of osteoporosis, is coupled to the ability of the peptide sequence to increase AC activity. The native h(PTH)-(1-34) sequence has been shown to have all of these activities. The hPTH-(1-34) sequence is typically shown as (A):
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His Asn Phe-OH (SEQ ID NO: 1) A PA0 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gin Asp Val-NH.sub.2 (SEQ ID NO: 2) B PA0 R-NH-R1-Val-Ser-Glu-Ile-Gln-Leu-R8-His-Asn-Leu-Gly-R13-R14-R15-R16-R17-R18- Glu-Arg-Val-R22-Trp-Leu-R25-R26-R27-Leu-Y (SEQ ID NO: 7) PA0 R-NH-Ser-Val-Ser-Glu-Ile-Gln-Leu-R8-His-Asn-Leu-Gly-Lys- His-Leu-Asn-Ser-R18-Glu-Arg-Val-R22-Trp-Leu-R25-R26-R27-Leu-Y (SEQ ID NO: 8) PA0 RNH-Ser-Val-Ser-Glu-Ile-Gln-Leu-R8-His-Asn-Leu-Gly-R13-His-Leu-Asn-R17-R18- Glu-Arg-Val-R22-Trp-Leu-R25-R26-R27-Leu-Y (SEQ ID NO: 9)
The following linear analogue, hPTH-(1-31)-NH.sub.2, has only AC-stimulating activity and has been shown to be fully active in the restoration of bone loss in the ovariectomized rat model (Rixon, R. H. et al (1994) J. Bone Miner. Res. 9, 1179-1189; Whitfield et al (1996), Calcified Tissue Int 58, 81-87; Willick et al, U.S. Pat. No. 5,556,940 issued Sep. 17, 1996):
The above molecule, B, may have a free carboxyl ending instead of the amide ending illustrated.
It is an object of the present invention to produce new PTH analogues with greater metabolic stability, increased bone restoration activity, increased AC activity, and minimal clinical side effects.